An in-depth characterisation of European seabass intestinal segments for assessing the impact of an algae-based functional diet on intestinal health

Sustainable farming of fish species depends on emerging new feed ingredients, which can alter the features of the digestive tract and influence animals’ overall health. Recent research has shown that functional feeds hold great potential for enhancing fish robustness by evoking appropriate responses at the intestine level. However, there is a lack of extensive and accurate descriptions of the morphology of the gastrointestinal tract of most farmed fish. We have characterised the intestine of European seabass thoroughly, by targeting four segments − anterior, mid, posterior and rectum. Results indicated that the anterior segment is mostly associated with absorption-related features; this segment has the largest absorptive area, the longest villi, and the highest number of neutral goblet cells (GC). The posterior segment and rectum have distinct histomorphometric features, but both seem to be important for immunity, displaying the highest count of acid GC and the highest expression of immune-related genes. The strongest proliferating cell nuclear antigen (PCNA) signal was observed in the anterior intestine and rectum, with PCNA+ cells appearing at the base of the villi and the corresponding villi branches. We have also evaluated the impact of a novel feed supplemented with a macro- and microalgae blend and found that there were no differences in terms of growth. However, the alterations observed in the mid intestine of fish fed the blend, such as thickening of the submucosa and lamina propria, an increased number of leucocytes, and higher expression of immune- and oxidative stress-related genes, suggest that algae may have an immunomodulatory effect. In the current article, we have described the morphology and expression patterns of the intestine segments of European seabass in detail and have presented a comprehensive report of the indices and methods used for the semi-quantitative and quantitative histomorphometric assessments, thereby providing useful information for future studies that aim to maintain intestinal health through dietary interventions.

climate change often triggers unwanted diseases that severely affect the economic sustainability of the sector 7,8 . The concept of One-Health has prompted the aquaculture industry to search for ingredients capable of promoting fish health and welfare and allowed functional feeds to gain traction 9,10 . Macro-and microalgae are valuable sources of bioactive compounds that can act as immune modulators 11,12 . Hence, algae are promising candidates for functional aquafeeds, with potential to boost the immune responses of farmed fish 13,14 . To thoroughly understand the functionality of such products, their impact at the intestinal level has to be delineated because this organ is the key site where diet evokes local immune responses 1 .
Integrated approaches, by combining histomorphometric indices and molecular biomarkers, can provide valuable information about the effects of novel feed ingredients on the morphology and functions of the intestine of farmed fish [15][16][17] . It should be noted, however, that morphologic traits can differ among species and along their intestinal tract, and studies on intestinal features still rely on traditional and laborious methods that largely vary among studies. The available literature commonly identifies three main intestinal segments in fish: anterior (or proximal), mid, and posterior (or distal) intestine 4,5,18,19 . But since the fish intestine does not have easily distinguishable segments like those in mammals, there is currently no consensus on its morphological division and nomenclature 20 , resulting in difficulties when comparing results across studies. For example, in studies on European seabass, the definition of the anterior and posterior intestines varies. Some define the anterior intestine as the segment from the pyloric caeca to the ileorectal valve, and the posterior intestine as the portion after this valve, i.e. the rectum 21 . Others considered the posterior section and rectum as distinct portions of the posterior intestine 18 . Furthermore, many studies fail to provide an accurate description of the intestinal portions used in their analysis, and the lack of a standardised terminology only adds to the confusion.
Current histological parameters-based determination of the fish intestinal health depends on classical morphological changes, for example diet-induced adverse effects such as villi shortening. Many of the studies that have reported such morphological changes have relied on semi-qualitative analyses [22][23][24] , but quantitative approaches at the intestine level are becoming increasingly popular 18,25 . Moreover, a previous functional analysis based on transcriptomic profiling attributed specific functions to the segments of the European seabass intestine. This analysis reported that the anterior and mid sections are mostly associated with feed digestion and nutrient absorption, whereas the posterior/rectal segments are especially relevant for immune functions 26 . The posterior/ rectal regions are also the preferred sections for understanding the diet-induced modulation of the microbial communities [27][28][29][30] . Nonetheless, the lack of a comprehensive study of the morphology and physiology of seabass intestine hampers the ability to selectively modulate specific targeted traits and hence contribute to precision nutrition.
We wanted to establish reference methods and guidelines for morphological studies at the intestine level, and we believe that this information can be used to assess European seabass intestinal health status, so that results can be easily compared with future studies. Hence, we performed an in-depth characterization of four segments of the seabass intestine (i.e., anterior, mid, posterior and rectum) using definable indices and following a holistic approach: (1) fast-track image analysis combined with traditional histology methods; and (2) evaluation of the gene expression patterns in relation to immunity, oxidative stress, and nutrient transport. These methods were then used to understand the impact of a functional diet supplemented with both micro-and macroalgae on the intestinal structure of European seabass. To analyse the histomorphometric traits, both quantitative and semiquantitative assessments were performed to compare the performance of these two commonly used approaches for evaluating the intestinal health of fish.

Materials and methods
Ethical statement. Fish rearing and sampling were conducted using routine and animal husbandry practices of commercial farming operations. The fish trial and experimental protocols were approved by the Ethical Committee of Riasearch Lda. (Murtosa, Portugal) overseen by the National Veterinary Authority (DGAV, Portugal), in compliance with the guidelines of the European Union (Directive 2010/63/EU) and Portuguese law (Decreto-Lei no. 113/2013, de 7 de Agosto) on the protection of animals used for scientific purposes. The study is reported in accordance with the ARRIVE guidelines.
The feeding trial was conducted in a saltwater recirculation system at the facilities of Riasearch Unipessoal Lda., Portugal. European seabass (initial body weight 118.6 ± 15.2 g) were randomly assigned to four 350 L fiberglass tanks (i.e., two tanks per dietary treatment) and fed the experimental diets for 8 weeks (54 days). Fish were hand-fed three times a day and at the end of every feeding cycle, apparent satiation of the fish was confirmed through visual observation. The system conditions were as follows: water temperature of 20 °C, salinity of 18‰, flow rate at 700 L/h (200%/h) and 12 h light/12 h dark photoperiod regime.
At the end of the feeding trial, and following a 48-h fasting period, 10 fish per tank were euthanised by anaesthetic overdose (MS222, 150 mg L −1 ). The different sections of the intestine were sampled, specifically the anterior region (i.e., right after the pyloric ceca), the mid intestine (i.e., the middle portion in between the anterior and posterior sections), the posterior region (i.e., right before the ileorectal valve), and the rectal section (i.e., right after the ileorectal valve). Samples were carefully washed and fixed in 4% formaldehyde (pH 7.0 ± 0.1) for histological evaluation or deep-frozen for gene expression analysis by RT-PCR. Sections were stained with Alcian Blue/Periodic Acid Schiff (AB/PAS) to detect the acid and neutral goblet cells (GC), and measure the villi length, the submucosa and lamina propria width, the muscularis thickness and the cross-sectional perimeter. (b) Sections stained with May Grünwald-Giemsa (MGG) were used to measure the microvilli height, and to count the lymphoid cells and granulocytes in the submucosa and lamina propria. Note the morphology of the different cells found in submucosa and lamina propria: granulocytes are big, ovalshaped cells, with pink granules in the cytoplasm and a blue-stained nucleus in the periphery; lymphoid cells are small cells that stain blue; and erythrocytes are elongated cells with a pink cytoplasm and centrally located blue-stained nucleus. (c) The number of acid and neutral GCs and the absorption area (total area occupied by the villi) were automatically determined by the software. All measurements were performed using the imaging software Olympus cellSens. See Suplementary Table S1 for more details about the quantitative analysis. Table 1. Description of the scores used to evaluate the intestinal morphology of European seabass -semiquantitative analysis. a Adapted from Urán et al. 50 , Silva et al. 49 and Penn et al. 48 ; b Adapted from Silva et al. 49 and Penn et al. 48 .

Score Description
Villi length and integrity a  www.nature.com/scientificreports/ (800 W) for 20 min. After cooling at room temperature, the slides were rinsed for 5 min in running tap water, and immunostained using the Novolink Polymer Detection System kit (RE7140-K, Leica Biosystems, Germany), according to the manufacturer's instructions. Thereafter the sections were incubated overnight with the primary antibody − anti-PCNA mouse monoclonal antibody (Santa Cruz Biotechnology, EUA) in a humidified chamber, at 4 °C. The antibody was diluted 1:2000 in 1% bovine serum albumin (BSA)/phosphate buffered saline (PBS). Counterstaining was performed with haematoxylin. The area occupied by PCNA + cells was automatically determined by the Olympus cellSens software, using the same procedures that were adopted for counting the GC ( Fig. 1c and Supplementary Table S1). Each MGG stained section was also semi-quantitively evaluated according to the criteria outlined in Table 1. To evaluate each intestinal segment's features, all samples were screened beforehand to ensure the accurate assignment of the score of 1, as morphologic traits vary throughout the tract. For example, in the anterior and rectum sections, a score of 1 indicates long, thin, well-organized villi with no tissue damage, while in the mid and posterior sections, it corresponds to medium-length villi with no tissue damage. Furthermore, as scores increase, so does the degree of alteration in comparison to score 1. This example demonstrates the importance of carefully assessing each section's specific morphologic traits to assign accurate and reproducible scores. Concerning the scores range, while villi length and integrity, submucosa thickness, and lamina propria width were assigned scores of 1-9, submucosa lymphoid cells, submucosa granulocytes, lamina propria lymphoid cells, and lamina propria granulocytes were assigned scores of 1-5. These scores were considered the most appropriate to define the different intrinsic intestinal segment's features.

Expression of genes in the different intestinal sections of European seabass.
Total RNA was extracted from the different intestinal sections (~ 30 mg; n = 16 per treatment), using TRIzol reagent (Invitrogen, USA) and NZY Total RNA Isolation kit (NZYTech, Portugal), according to the method described by Ferreira et al. 31 . The quantity and purity of the extracted RNA were spectrophotometrically evaluated, while RNA integrity was assessed using an agarose electrophoresis gel as previously described 17 . Using 1 μg of RNA, first strand cDNA was synthesized, and real-time (RT) PCR assays were performed on a CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, USA) with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories, USA). Following the protocol described by Ferreira et al. 17 , reactions were performed in duplicate. The employed thermal cycling conditions were: 95 °C for 30 s, followed by 35 cycles of two steps of 95 °C for 5 s and 60 °C for 30 s. The specificity of the RT-PCR reaction was ensured by a post-amplification dissociation curve, while PCR efficiency for each gene was determined using a fivefold serial dilution of cDNA from all the samples used in the experiment. A total of 22 genes (four reference genes and 18 target genes) were analysed, including genes associated with a) immunity (i.e., igM, immunoglobulin M; cd4, cluster of differentiation 4; il-6, interleukin-6; il-8, interleukin-8; il-1β, interleukin-1 beta; tnf-α, tumor necrosis factor-alpha; tcrβ, T-cell receptor antigen receptor beta chain; tlr9, toll-like receptor 9; casp3, caspase 3; pcna, proliferating cell nuclear antigen; pisc1, piscidine 1); b) oxidative stress (i.e., gpx, glutathione peroxidase; sod, superoxide dismutase; cat, catalase); and c) nutrient digestion and absorption (i.e., alp, alkaline phosphatase; malt, maltase; fabp2, intestinal fatty acid binding protein; aqp1, aquaporin 1). The primer sequences, accession numbers, annealing temperatures, and PCR efficiencies can be found in Supplementary Table S2. The stability of the reference genes (i.e., 18 s, β-actin, ef1α and gapdh) was determined by the geNorm algorithm 32 , within the qbase + software, version 3.2 (Biogazelle, Zwijnaarde, Belgium-www. qbase plus. com), and a normalization factor was calculated for each sample. The comparative critical threshold (ΔΔCT) method 33 was used to calculate the relative abundance of the target genes, as described by Ferreira et al. 17 .
Statistical analysis. The IBM SPSS software platform 27.0 (SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis. The assumptions of normality and homogeneity of variances were checked by Kolmogorov-Smirnov and Levene's tests, respectively. Data were transformed when necessary. For comparisons between intestinal sections, one-way ANOVA followed by Tukey's multiple comparison test was performed. The level of significance used was P < 0.05. Independent samples t-test was applied to understand the differences between the two diets. A Spearmen's rank correlation coefficient test was applied to the histology-based variables of the quantitative and semi-quantitative data. Significant correlations were considered at the bilateral levels of 0.05 or 0.1. The principal component analysis (PCA) was performed using XLSTAT version 2022.1.2. (Addinsoft, USA) to differentiate the samples from the different study groups.

Histomorphology of the different sections of the intestine of European seabass. Quantitative
analyses were performed to describe the micromorphology of the different intestinal sections. The results of the analyses are presented in Table 2 and the differences in the morphology of anterior, mid, posterior, and rectal sections are evidenced in Fig. 2.
The anterior region had the largest absorptive area, the longest villi, the thinnest submucosa, and the largest total area of PCNA + cells compared to all other sections. The posterior and rectal sections had higher GC and acid GC counts than the anterior and mid regions. The average area of the GC (including acid and neutral cells) was also significantly larger in these sections (i.e., posterior intestine and rectum). As for the GC and acid GC counts expressed per intestinal absorptive area, the highest values were for the posterior intestine, followed by the mid intestine and rectum, with the anterior intestine presenting the lowest value in comparison with all other sections. On the other hand, the anterior section had the highest count of neutral GC compared to the mid and posterior regions. However, when such neutral GC counts were expressed per absorptive area, we did not detect any significant differences between sections. The anterior and mid regions had significantly higher number of Concerning the PCNA + cells per absorptive area, the anterior intestine presented the highest values, but was not significantly different from the rectum. Moreover, as observed in Fig. 2, the PCNA signal in the anterior Table 2. Histomorphology of the different intestine sections of European seabass using a quantitative analysis. Values presented as mean ± SD (n = 20 samples per section). One-way ANOVA followed by Tukey's multiple comparison test was performed. Different letters denote significant differences between intestinal sections. PCNA, Proliferating cell nuclear antigen. Figure 2 provides a visual representation of the characteristics of the distinct sections.   www.nature.com/scientificreports/ portion was detected in the basal part of the complex folds and in the villi branching points, while in rectum the PCNA + cells were concentrated at the basis of the villi. A semi-quantitative analysis with well-defined and validated scoring system is described in Table 1 and was applied to the different intestinal sections in order to describe the intrinsic intestinal segment's features (Supplementary Table S3). Likewise, the quantitative analyses, the semi-quantitative approach confirmed that the anterior intestine is characterized by long villi with very few irregular folds and a thin submucosa layer. The mid and posterior segments had medium sized villi with few irregular folds, and the rectum had a very thin lamina propria with very few granulocytes. It is important to note that the attributed scores are specific to each section and cannot be used to compare differences between them.
Gene expression patterns of the different sections of the intestine of European seabass. A panel of 18 genes, involved in immunity, oxidative stress and nutrient digestion and absorption, were selected to determine the gene expression patterns associated with the different sections of the intestine of European seabass (Fig. 3). The expression of genes involved in the different immune pathways was, in general, higher in the rectum. Specifically, the mRNA levels of il-8, il-1β and tlr9 were significantly higher in the rectum compared to all other sections. The genes cd4, tnf-α, and tcrβ had higher expression in rectum only in comparison with the anterior and mid regions. In the rectum, the expression of igM and casp3 was higher compared to only the mid intestine. The expression of il-6 was higher in both the anterior and rectal section but compared to only the mid segment. In contrast, the pcna gene was found to have the highest transcript levels in the anterior region, www.nature.com/scientificreports/ followed by the rectum, with the mid region presenting the lowest expression values. There were no significant differences between the expression of the pisc1 gene in the different intestinal sections (Fig. 3a). The relative expression of genes associated with oxidative stress (i.e., sod and cat) differed significantly between the intestinal sections. The genes sod and cat were significantly downregulated in the rectum compared to all the other regions. The intestinal sections had similar levels of the mRNA of the gene gpx (Fig. 3b).
Concerning the genes related to nutrient digestion and absorption, the highest expression of the malt gene was observed in the anterior region, followed by the mid and posterior intestine, with the rectum presenting the lowest expression of this gene. While the fabp2 gene was significantly lower in the rectum compared to all the other sections, the expression of aqp1 was lower in the anterior region compared to the mid, posterior, and rectal sections. We did not observe any significant differences between the expression pattern of the alp gene along the intestinal tract (Fig. 3c).
Principal component analysis -morphology and functionality of the different intestinal sections of European seabass. Principal component analysis (PCA) was employed to visualise all the variables analysed in the anterior, mid, posterior, and rectal portions of the intestine of European seabass. The different intestinal sections were separated sequentially (as in the intestine) along the F1 axis, with the first two dimensions of the PCA biplot explaining 46.34% of the variability in the experimental data (Fig. 4). The variables absorptive area, villi length, expression of the genes malt, fabp2, sod and cat, cellular proliferation parameter (PCNA + cells), granulocytes in submucosa and lamina propria, and lymphoid cells in lamina propria were found to be associated with the anterior intestinal section. The immune-related genes, muscularis thickness, microvilli height, acid GC, submucosa width, and expression of the genes aqp1 and alp appeared in the quadrants where the posterior and rectum samples clustered in the PCA plot. The mid samples were in a different quadrant than the other sections (F1 positive/F2 negative).
Growth performance of European seabass fed the experimental diets. After an 8-week feeding period, there was no significant difference in final body weight, weight gain, specific growth rate (SGR), and feed conversion ratio (FCR) of European seabass that were fed with a diet containing 2% of a commercial algae blend composed of Gracilaria sp., Nannochloropsis sp. and Aurantiochytrium sp. compared to those fed with a CTRL diet (Fig. 5).

Impact of the algae blend-supplemented diet on the intestine of European seabass.
To understand the impact of the algae blend-supplemented diet on the intestine of European seabass, both the histo- www.nature.com/scientificreports/ morphology and gene expression patterns were analysed in the fish fed the CTRL and the Blend diets. Overall, the algae blend had very limited impacts on the morphology and functionality of the intestine, with most of the observed differences being located in the mid intestine (Table 3 and Supplementary Figure S2). A reduction in the villi length, number of GC and acid GC, and the total area occupied by the GC was observed in the mid intestine of fish fed the Blend diet compared to those fed the CTRL diet. The mid intestine of the fish fed the blend had thicker submucosa and wider lamina propria, both parts with a significantly higher number of lymphoid cells, and submucosa with more granulocytes. The area occupied by the PCNA + cells per absorptive  www.nature.com/scientificreports/ area in the mid intestine was also significantly increased after the blend diet feeding ( Table 3). Alterations of the gene expression patterns were restricted to the mid region, with an upregulation of the il-8, pisc1 and gpx genes in fish that consumed the algae-supplemented diet (Supplementary Figure S2). The differences observed in the other intestinal sections, in response to the blend diet, were limited to a higher number of lymphoid cells counts in the submucosa of the rectum, and higher number of granulocytes in the submucosa of the anterior and rectal sections (Table 3). A PCA biplot created using all the analysed variables in each intestinal section could not discriminate between the CTRL and Blend samples (Supplementary Figure S3). On the other hand, when only the variables significantly affected by the experimental diet in the mid intestine were selected and visualised in a PCA biplot, the CTRL and Blend samples were separated along the F1 axis, with the first two dimensions explaining 60.64% of the variability of the data (Fig. 6). The blend samples were associated with immune-related parameters such as expression of il-8 and the antimicrobial peptide pisc1, the presence of the lymphoid cells in submucosa and lamina propria, and the presence of granulocytes in submucosa. The lamina propria width and submucosa thickness and the oxidative stress-related gene gpx were also found in the F1 positive quadrant, where the blend samples were mostly clustered. The variables villi length, GC, acid GC, acid GC per absorptive area and total area occupied by the GC were positioned on the F1 negative quadrant and seemed to be associated with the control samples.
Quantitative versus semi-quantitative approaches for evaluation of the intestinal health. The experimental diet-induced alterations of the intestine histomorphology were evaluated both quantitatively ( Table 3) and semi-quantitatively (Table 4). In general, the differences captured by the quantitative assessment were also reflected in the semi-quantitative approach-based results, namely the increase in the submucosa thickness and lamina propria width, higher number of lymphoid cells in the submucosa and lamina propria, and granulocytes in the submucosa of the mid intestine of blend-fed fish. But some differences detected through the quantitative approach could not be perceived by the semi-quantitative analysis, namely the effects of the blenddiet on the villi length in the mid intestine, the increase in the lamina propria width in the anterior intestine and the rise in the number of granulocytes in the submucosa of the posterior intestine. Nevertheless, we observed a significant correlation between the variables of all the intestinal sections analysed quantitatively and semiquantitatively, i.e., villi length, submucosa thickness, and lamina propria width, and presence of leucocytes in the submucosa and lamina propria (Supplementary Figure S4).

Discussion
The production and economic viability of the aquaculture sector are adversely impacted by the rising incidence of disease outbreaks in fish farms 7 . Functional feeds are being recognised as promising tools to enhance fish robustness 10 , a key factor for boosting the profitability of the aquaculture sector. Gaining an in-depth understanding of the morphology and functional potential of the gastrointestinal tract is crucial for comprehending the functionality of novel feed components and allow comparison of results among studies. The intestinal health of www.nature.com/scientificreports/ farmed fish is intimately related to both nutrition and immunity 1 , both of which can affect the morphology and functions of the intestine. To advance the field of fish intestinal health, we conducted a comprehensive characterisation of the European seabass intestine and propose specific indices and detailed scoring system that were correlated with intestinal functionality. In a previous study, Verdile et al. 5 performed a detailed examination of the intestine of rainbow trout (Oncorhynchus mykiss), exploring different sections in specimens of varying weight ranges. Although growth-induced modifications of the intestine's morphology were minimal, marked disparities were noted between the different sections of the intestine. Previous studies on European seabass intestine have only examined the anterior 25 , posterior/rectal segments 18 , or both the anterior and posterior segments 19 . In contrast, our study provides a comprehensive analysis of the four distinct sections of the European seabass intestine − anterior, mid, posterior, and rectum. The histomorphological parameters, gene expression patterns, and the position of the associated clusters in the PCA plot indicate the distinct morphology, functionality, and location of the different segments of the intestine. The samples collected from the anterior segment were linked to parameters associated with the processes of digestion and absorption. This segment exhibited the largest absorptive area and the longest villi, as well as the thinnest submucosa and highest count of neutral GC. In fish, the intestinal mucus layer produced by the GC is thought to play different roles, ranging from lubrication, absorption and immune defence 34 . The neutral GC, in specific, have been previously linked with improved digestion and absorption in European seabass 19 . The gene patterns observed in this segment further corroborate the importance of the anterior intestine within the digestive tract of fish. The feed efficiency of fish is highly dependent on both the digestive enzymes and the proteins involved in nutrient transportation processes 35 . In the present study, the involvement of anterior segment in the carbohydrates and lipid digestion and absorption is highlighted by the high expression of the genes encoding for the carbohydrate enzyme maltase (malt) and the fatty acid-binding protein 2 (fabp2) observed in this section. Therefore, the increased surface area observed in the anterior intestine in comparison with the other segments, coupled with the highest counts of neutral mucin-secreting cells, and high expression of genes that encode for digestive and transport proteins indicate that the region has the ideal environment for both nutrient digestion and absorption, in accordance with previous reports 26 .
The rectum and posterior intestine samples were found to be associated with immune-related variables. We observed the highest expression of immune-related genes in the rectum, followed, in general, by the posterior section. Moreover, the highest number of acid GC, that aid in protection against invading pathogens 36,37 , were found in the posterior/rectal sections, highlighting the importance of these segments for the intestinal immunity. A previous study examining in European seabass with a final body weight of 59 g revealed morphological differences between the posterior intestine and the rectum 18 . Our results show that the rectum of European seabass has longer villi compared to the posterior intestine and the posterior intestine has a higher density of GC, corroborating the findings of Torrecillas et al. 18 . In contrast to this study, we observed no variation in submucosa width between the posterior intestine and rectum, despite the latter having more lymphoid cells in the submucosa. These differences could be attributed to variations in fish body size (i.e., 60 vs 200 g in the present study) or exposure to different environmental challenges that might affect leucocyte infiltration. Our study compared four separate segments along the intestinal tract and revealed marked morphological differences in the rectum compared to the other three sections. Specifically, this region displayed the thickest muscularis, narrowest lamina propria, and longest microvilli. The thick muscularis layer observed in rectum suggests that intestinal motility 38 is crucial in this section, potentially for the release of waste. In addition, the longer microvilli of the rectum indicate that some important absorption processes still occur in this segment. When examining the gene expression patterns, it appears that the rectum is primarily involved in water absorption, rather than nutrient digestion, as compared to the other segments. As one of the main osmoregulatory organs in marine teleost, the intestinal epithelium plays a key role in water transport-processes through the action of membrane proteins, such as aquaporins 39 . Similarly to findings reported by Giffard-Mena et al. 40 , the mid, posterior, and rectal segments of the intestine seem to be Table 4. Histomorphology of the different intestine sections of European seabass fed the control or the blend diets using a semi-quantitative analysis. CTRL -fish fed the control diet, Blend -fish fed the blend diet. Scores 1-9 were assigned to villi length/integrity, submucosa width, and lamina propria width; while scores 1-5 were assigned to denote the presence of leucocytes (i.e., lymphoid cells and granulocytes) in the submucosa and lamina propria. Values presented as mean ± SD (n = 10 samples per dietary treatment), based on a semiquantitative analysis. Independent samples t-test was employed to detect significant differences between the groups within each intestinal section. Different letters denote significant differences between dietary treatments. www.nature.com/scientificreports/ more relevant in the salt-water interactions across the intestinal barrier in European seabass, as evidenced by their high expression of aqp1 compared to the anterior section. The expression levels of the intestinal brush border membrane enzymes herein studied were segment-specific, except for alkaline phosphatase (alp). This enzyme displayed a consistent expression pattern across all the intestinal segments. The intestinal alp has been recognised for its role in preventing inflammation, both in the gut and at the systemic level 41 . According to the PCA plot, the variables malt and fabp2 were found to be associated with the anterior section, along with other digestion/absorption-related parameters. In contrast, the expression of the alp gene appeared in the quadrants where the posterior and rectum samples clustered, in association with immune-related variables. Therefore, the ubiquitous expression of the alp gene throughout the intestine may play a role in maintaining intestinal homeostasis, which supports previous findings 42 .
The selected gene expression patterns were unable to differentiate between the mid and posterior regions. Nonetheless, several histomorphometric indices, such as the absorption area, microvilli height, number of GC and acid GC, area of GC, and quantity of leucocytes in the submucosa and lamina propria, were able to distinguish these regions. While the posterior intestine samples were positioned in the same quadrant of the PCA biplot as the rectum samples, the mid intestine samples clustered in a specific area of the PCA biplot, and without any clear association with the assessed variables. The mid intestine seems, therefore, to be an intermediate region for both absorption and immune-related processes.
In order to understand the turnover of the enterocytes in different sections of the intestine, we analysed the localization and distribution of the proliferating cell nuclear antigen (PCNA) by immunostaining positive cells. The anterior portion of the intestine, followed by the rectum, had the strongest PCNA signalling, although there were differences in localization. Similar to what was observed in rainbow trout 5 , the PCNA signal was detected both at the basis of the villi and at the villi branching points in the anterior intestine of European seabass, while in the rectum, PCNA immunolocalization was restricted to the base of the folds. The results suggest that the stem cell zone is most likely located at the basal part of the villi. This is consistent with previous findings in rainbow trout by Verdile et al. 43 , who found that the sox9 + cell zone (a marker for stem cells) in the intestine was similar to the PCNA + cell zone. The understanding of the stem-cell population's localization in the gastrointestinal tract is crucial for the development of novel in vitro tools, such as organoids, for the study of fish intestine. Organoids have recently emerged as one of the most promising tools for studying the intestinal health of farmed animals 44 .
Fish nutrition studies should accurately report the impacts of feeds on the intestinal health, and for this it is important to precisely describe the changes in the intestine segment selected for the analysis. Based on our findings, we propose that the intestine of European seabass can be divided into four different segments with unique morphological and/or gene expression profiles: 1) the anterior intestine, 2) the mid intestine, 3) the posterior intestine, and 4) the rectum. It is important to note that the length of the intestine can vary with fish size, and therefore, we recommend a standardized method for identifying and sampling these segments. Specifically, the anterior intestine should be sampled immediately after the pyloric ceca; the mid intestine should be sampled from the middle region between the pyloric ceca and the ileorectal valve, the posterior intestine should be the portion preceding the ileorectal valve, and the rectum should be the segment immediately after the ileorectal valve. Using this standardized approach for sample collection and nomenclature for intestine segments will ensure consistent and accurate reporting of feed impacts on intestinal health and allow comparison with future studies.
After conducting a detailed study on the different intestinal segments of European seabass, we aimed to investigate the effects of a novel macro-and microalgae blend on the intestinal functionality of the fish. Although the dietary inclusion of the blend did not affect fish growth performance or feed utilization parameters, the results suggest that the diet can alter the intestine morphology. Most studies that evaluated fish feed utilization and immune status relied either on the anterior intestinal segment 19 or on the posterior/rectal segment 18 , respectively. However, in this study, the subtle differences between the CTRL and the Blend diet were mostly perceived in the mid intestine. When the impact of the blend was evaluated in the different intestinal segments using either a quantitative or semi-quantitative approach, the mid section presented, in general, the highest response towards the diet, which may indicate that this segment is more sensitive to external stimuli. The feeding trial lasted for only 8 weeks, hence the observed villi shortening and the reduced number of acid GC in the mid intestine are likely to be an adaptive response to the diet. Further evaluation of such alterations in the intestine structure is required after a longer-term feeding trial. Nonetheless, some immunomodulatory effects were already observed in various intestinal sections, including thickening of the submucosa (mid intestine), widening of the lamina propria (anterior and mid sections), an increase in the number of lymphoid cells in the submucosa (mid and rectum segments), granulocytes in the submucosa (all sections), and lymphoid cells in the lamina propria of the mid intestine. These observations, along with the increased expression of immune-and oxidative stress-related genes (il-8, pisc1 and gpx), in the mid region, point towards an immunomodulatory effect promoted by the algae blend. This potential functionality should be further explored after a disease challenge, such as a bacterial infection. However, it is important to note that experimental conditions, namely temperature and salinity, can influence gut structure and functionality and yield varying outcomes [45][46][47] . In a previous study, we observed that European seabass fed a micro-and macroalgae blend and reared under similar temperature conditions as the present trial (21 °C), but with higher salinity levels (35‰), had an enhanced mucosal immune response upon infection with Tenacibaculum maritium 17 . Overall, these results suggest that dietary supplementation with algae could be a promising strategy to improve the robustness of European seabass.
To evaluate the impact of feeds on fish intestinal health, researchers have often conducted a semi-quantitative/ score-based analysis focused on a determined intestinal segment. Such scoring system commonly used in literature often lacks clear definition and description of the selected indices and can lead to unreproducible results 48,49 . Moreover, due to the lack of a well-defined and validated scoring system, it is more likely for untrained individuals to make errors and have unreproducible results 20 . As morphologic traits vary throughout the tract, it is crucial to assess each section's specific morphologic traits and all samples beforehand to ensure the accurate assignment www.nature.com/scientificreports/ of the score. Hence, our study provides a detailed description of a set of scores for the semi-quantitative assessment of several morphological parameters of the intestine such as 1) villi length and integrity; 2) submucosa thickness; 3) lamina propria width; 4) submucosa lymphoid cells; 5) submucosa granulocytes; 6) lamina propria lymphoid cells; and 7) lamina propria granulocytes. The proposed scoring system includes validated and clearly defined criteria for each intestinal section, allowing researchers to select a scoring method that generates reliable and consistent data. Alternatively, recent studies investigating the morphology of the intestine have utilized quantitative approaches to produce precise measurements [18][19][20] . In the present work, we have also developed a detailed description of the indices and methods used for the quantitative assessment, which also allow direct comparisons among intestinal segments. We have used an image analysis software to automatically estimate the absorptive area, count the GC, and evaluate cellular proliferation (determined by the total area occupied by PCNA + cells). For all other parameters, we have employed a semi-automatic approach. The quantitative assessment method enables us to measure the same parameters that are commonly evaluated through a score-based analysis (e.g., villi length, submucosa and lamina propria widths, and leucocyte infiltration) 20,48-50 , as well as several other additional parameters such as total absorptive area, muscularis thickness, and total GC counts in a cross-section. This approach has enormous potential to further evolve into a completely reliable automatic image analysis using artificial intelligence technologies for fast-track evaluation of fish intestinal health.
Although the semi-quantitative analysis is a less time-consuming and relatively easier method to assess the intestine histomorphology, it relies on pre-defined scores, which can increase bias 51 . In addition, unlike the quantitative assessment, the semi-quantitative analysis requires highly trained and experienced observers 20 . In the present study, we found a significant correlation between the values obtained by both quantitative and semi-quantitative analyses for the parameters evaluated within each intestinal segment. This indicates that both methods are valid for assessing intestinal health. While the semi-quantitative analysis may be useful for a quick pre-assessment, it is important to define the scoring system correctly to ensure good repeatability. However, we prefer the quantitative approach overall, as it allows for more easily comparable measurements across studies. To ensure robust and reproducible data from both methods, it is crucial that studies provide a detailed description of the methodology for each index in the methodology section.
In conclusion, by providing an in-depth characterisation of the different segments of the intestine (i.e., anterior, mid, posterior, and rectal) of European seabass, the present study provides valuable information about the intestinal morphology. Overall, we found that histological characteristics and gene expression profiles were specific to each intestinal segment. The anterior region appears to be primarily associated with absorption processes, with the largest absorptive area, longest villi, and a high expression of genes encoding digestive and transport proteins. In contrast, the rectum, followed by the posterior intestine, displayed the highest expression values for immune-related genes, highlighting their importance for mucosal immunity. The thick muscularis and long microvilli observed in rectum, coupled the high expression of the aqp1 gene, most likely indicates that both waste release and water absorption are also important processes occurring in this segment. Finally, the mid intestine appears to be a transitional region for both absorption and immunity. Hence, the generated knowledge can assist in arriving at better decisions regarding the choice of the appropriate segment and morphometric indices for future experiments to study nutritional modulation and its effects on intestinal health. Furthermore, transition from a semi-quantitative to a quantitative analysis of intestine histomorphology is crucial for adopting digital technologies that produce more precise and fast histology-based results. As a futuristic perspective, the quantitative analysis offers a promising avenue for machine learning approaches in this field, allowing for high throughput image analysis of intestinal histological sections using artificial intelligence.